弓形虫胚层发育相关蛋白的原核表达及免疫原性研究

Study on Prokaryotic Expression and Immunogenicity of Toxoplasma gondii Embryogenesis-related Protein

试验旨在研究弓形虫胚层发育相关蛋白(TgERP)的免疫原性,以弓形虫RH株的基因组DNA为模板,扩增TgERP基因,将其连接于表达载体pET-30a(+)后,转化至Escherichia coli BL21(DE3)感受态细胞进行IPTG诱导表达,应用Western blotting对重组蛋白的反应原性进行分析,并用纯化后的TgERP蛋白免疫新西兰大白兔,制备多克隆抗体。结果显示,在37℃条件下用1.0mmol/L IPTG诱导6h表达可溶性TgERP蛋白的量最大。SDS-PAGE结果显示,目的蛋白分子质量为16.7ku,以可溶形式表达,纯化后蛋白条带单一。经Western blotting分析,TgERP蛋白有较好的反应原性;制备的多克隆抗体效价较高,可达1∶51200,表明该蛋白具有较好免疫原性。提示,TgERP蛋白可作为血清学诊断方法的候选抗原和弓形虫病疫苗的候选分子,为建立弓形虫新型诊断方法和研制新型弓形虫疫苗奠定了基础。

In order to analyze the immunogenicity of Toxoplasma gondii embryogenesis-related protein （TgERP）, the TgERP gene was amplified from genomic DNA of T. gondii RH strain by PCR and then cloned into prokaryotic expression plasmid pET-30a （＋）. The recombinant plasmid pET-30-ERP was transformed into Escherichia coli BL21 （DE3） and induced by IPTG. The expressed product was purified by Ni-NTA His Bind Resin affinity chromatography and analyzed by Western blotting. The polyclonal antibody against the recombinant protein was prepared by immunizing New Zealand White rabbits with the purified protein. PCR amplification and restriction analysis proved that the restriction prokaryotic expression plasmid pET- 30a （＋） was constructed correctly and sequencing results showed that the cloned TgERP gene was identified to be the corresponding sequence reported on the website （http： //toxodb. org）. The TgERP protein was expressed under 1.0 mmol/L IPTG at 37℃, shaking for 6 h. SDS PAGE analysis showed the protein product was successfully purified with a molecular weight of about 16.7 ku. Western blotting and ELISA analyses indicated that the TgERP protein reacted with sera from vaccinated rabbit. In conclusion, it could be a potential candidate antigen for developing new detection methods or new sub-unit vaccine against toxoplasmosis.